Outline

Objective: In recent years brain tumor initiating cells have become one of the major targets for future therapies. Individualized therapies tailored to the eradication of this specific subgroup of cells may become possible in the future. Isolation of homogenous cell populations from human brain tumor tissue is a prerequisite for the exact characterization of tumor cell types, the analysis of cancerogenous potential and the development of future therapies.

Methods: Here we present optimized dissociation protocols that generate single cell suspensions from human glioblastomas. Manual mechanical dissociation causes fluctuations in the yield of viable cells due to pipetting at different speeds. In order to increase the reproducibility of enzymatic dissociation and to simplify the experimental procedure, we developed a semi-automated protocol for brain tumor dissociation.

Results: Neural tissue dissociation using papain or trypsin in combination with gentle dissociation results in more than 1E07 viable cells per 100 mg tissue. In contrast, Collagenase D is not suitable for this purpose as it yields a lower number of viable cells.

Single cell suspensions from glioblastoma samples often contain large quantities of myelin debris, which considerably impairs immunostainings and cell isolation. We show that a myelin removal protocol efficiently removes myelin debris and thereby improves immunostainings and magnetic cell isolation (MACS). Besides myelin debris, a high number of erythrocytes is undesirable e.g. for cultivation experiments or FACS. We demonstrate that it is possible to remove myelin debris and erythrocytes simultaneously from single cell suspensions

Conclusions: Taken together, these protocols accomplish a high yield of viable cells, the conservation of antigen epitopes for immunostainings and cell isolation experiments, the removal of myelin debris and reproducible standardization of the procedure